Multiple use of antibiotics

In the feature article “Intracellular Activity, Potential Clinical
Uses of Antibiotics” Robert M. Rakita (ASM News, 64, 570, 1998)
discusses the three-way interaction of the pathogens, host defense
cells, and antimicrobial agents, especially inside the neutrophils
and macrophages. The preparation of newer macrolide and
quinoline antibiotics try to achieve higher intracellular levels with
greater antimicrobial activities and with minimal cellular damage.
The goal being the control and elimination of the infecting bacteria.
What is often over-looked is the broad cellular reactions of
antibiotics in addition to their antimicrobial and clinical response.
Inhibiting a pathogen’s growth with antibiotics usually includes
the inhibition of cellular protein synthesis while its elimination
depends on its intracellular location and the host’s immune
responses. Many more conditions can effect the reactivity of
pathogens and antibiotics in the complex host tissues than in the
controlled in-vitro Tissue Cell Cultures. The variable tissue
pathogenicity also contributes to the variable antibiotic sensitivity
requiring adjustments for each pathogen and their tissue
location.(1)
Prior to the availability and application of antibiotics for the
control of diseases, gold salts, arsenicals, sulfa drugs and other
various chemicals were used to ward off the offending bacterial
pathogens without killing the patient. The clinical application of
antibiotics started in the early ‘40’s when penicillin became
available during WWII as the first miracle drug from a penicillium
mold. A few years later when the broad spectrum tetracycline
antibiotics became available their clinical use spread like a gold
rush. The bacterial sensitivities and potential clinical application of
the new antibiotics were extensively used by clinics and
laboratories. The choice of antibiotics was made after the isolation
and identification of the infecting agent. The development of
allergies and toxicities to some antibiotics limited their use in some
patients. Microcidal penicillins inhibit bacterial cell wall synthesis,
whereas the tetracyclines (macrolides) are micro static inhibiting
protein synthesis and the growth of the wall-less bacteria such as
mycoplasmas.
The initial use of high dosage antibiotics in some chronic
disease patients may cause a flare or clinical worsening with a
serologic rise in antibody titer to a suspected microbial agent such
as mycoplasmas. A temporary flare of symptoms following
antibiotic treatment is often referred to as a Jarisch Herxheimer
reaction. The flares often occur in joints or areas that have been
quiet or dormant since the arthritis was first observed. Knowing
this the patients are encouraged by the temporary worsening
following their antibiotic treatment. The delayed reaction resulting
from the release of microbial antigen into the sensitized host tissue
as in a “Graft vs. Host” reaction that is not a drug sensitivity.
Similarly the occurrence of physical &/or mental stress could also
initiate clinical worsening with a rise in microbial antibody titer.
The flare reaction could also result from the released microbial
antigen complexing with its circulating antibodies to promote
Complement Fixation. The antibiotics, tetracyclines, can also act
like the immunosuppressant steroids by blocking the formation of
the antibiotic+antigen complex that initiates inflammation. Many
clinical disorders are considered Immune Complex Diseases of
infectious origin, such as rheumatoid arthritis and Lupus, resulting
from the activation of complement and proteolytic destruction of
tissues with the deposition of Immuine Complex on the kidneys
and other tissue cell membranes.
The tetracycline antibiotics are potent metal chelating,
complexing, agents and comparable in action to the clinical use of
the chelating agents ethylenediaminotetraacetate, EDTA, and
penicillamine. Consequently the mode of antibiotic administration,
Intravenous or Oral (between meals), could have an affect on
the composition of their absorption state and thus their reactivity.
When complexed with divalent trace metals (Cu, Zn, Mg, Se, etc.)
The antibiotics become antioxidants or electron scavengers. As
such the metal antibiotic complex becomes antiinflammatory
neutralizing free oxygen radicals. By combining with metaloproteins
and metaloenzymes such as collagenase, antibiotic therapy can
inhibit collagen tissue destruction. If used excessively in high doses
the antibiotics, as protein synthesis inhibitors, could also inhibit the
synthesis and function of essential cellular proteins and not just the
pathogens.
Because of their immunosuppresive actions the macrolide
antibiotics can block and limit the immune complex (Antibody +
Antigen) formation and thus stop the complex induced
inflammation. In cases with low pathogenic activity, such as
mycoplasmas, pulsed antibiotic therapy with lower doses over
longer periods has proven more effective and with fewer side
effects. Tissue cells will survive intermittent (pulse) treatment of
tetracyclines but not constant exposure even at lower doses. In the
chronic immunologic disorders of probable infectious etiology
high daily antibiotic doses are not essential or effective for the less
virulent agents.
Bioassays for antibiotic levels in blood and tissues measures
the antimicrobial action that would not explain their other activities
based on intracellular concentration.
Although suspected of infectious origin the clinical trials of
minocycline antibiotic in rheumatoid arthritis was based primarily
®
Medical data is for informational purposes only. You should always consult your family physician, or one of our referral physicians prior to treatment.
on its inhibitory action of the metalloenzyme, collagenase, that
destroys the connective tissues and joint cartilage causing the
inflamed joints.
The effectiveness of treatment with minocin antibiotic was
based primarily on the eradication of arthritis inflammation rather
than infectious agents. The maximum effectiveness of the
antibiotic treatment was found in the duration of therapy indicating
a slow healing process that has to balance cell growth versus
inhibition of protein synthesis and microbial growth by the
multiple antibiotic actions. Growth inhibiting antibiotics may
control mycoplasma or microbial growth for an indefinite period
until the neutralizing antibodies and immune system process their
elimination.
Antibiotics can be used in the identification of the infectious
bacterial agent(s). In cases where the agent can not be isolated and
identified or the DNA can not be matched it is possible that
antibiotic therapy will cause the release of the microbial antigen to
initiate a specific antibody response. The serologic measure of a
change or response in the serum antibody level to a bacterial
infection would indicate its presence. The sero conversion or the
increase in antibody titer, resulting from the administration of a
vaccine would indicate the host’s immune responsiveness to a
particular antibiotic therapy. The specificity and sensitivity of the
serologic response depends on the test used, such as: growth
inhibition, neutralization, agglutination (ELIZA), complement
fixation, immunoblotting.
A rise in serum antibody level during the acute to convalescent
phase on antibiotic therapy would indicate a concurrent infection or
the antigen release from a persisting silent infection. A similar
positive sero conversion with a rise in antibodies could be observed
in a patient following physical or mental stress. No rise in antibody
titer to a vaccine, infection or stress would indicate an
immunodeficient agammaglobulinemia subject with limited
antibody production and immune defence. In rheumatoid arthritis
and other infectious diseases that initiate the anti-antibody response
(rheumatoid factor) RF the antibody levels are inversely related
causing an apparent decrease or negative sero conversion.
Following antibiotic treatment when the mycoplasma antibody
level increases, the RF test results will be lower.
The use of generic antibiotics may have the same
antimicrobial potency while their systemic action in the host may
varay significantly. For example in the treatment of RA the generic
minocycline is reportedly less effective than minocin. In some
patients this difference in antibiotic action could result from patient
differences.

Modification of the antibiotic olivomycin

The aureolic acid family of antitumor antibiotics includes a group of
clinically active agents such as olivomycin I (olivomycin A), mithramycin,
and also chromomycin A3 and durhamycin.1 The antibiotics of
the aureolic acid family interact with the DNA minor groove in high-
GC-content regions in a nonintercalative way and with the requirement
for Mg2+ ions.

The antitumor antibiotic olivomycin I was discovered at the Gause
Institute of New Antibiotics, Moscow.3 Comparative study of the
antitumor action of olivomycin I and chromomycin A3 in in vivo
experiments on murine lymphosarcoma LY01 revealed that the chemotherapeutic
index (LD50/DIT50) of olivomycin I is more favorable
(2.35) than that of chromomycin A3 (0.99). A similar study on the
inhibitory effect of olivomycin I, chromomycin A3 and mithramycin
against transplantable murine leukemia La showed that a similar
antitumor effect (an increase in the lifespan of mice by 25%) can be
achieved at lower doses of olivomycin I than those for the other
aureolic acid antibiotics studied. Clinical investigations of mithramycin
and olivomycin I showed that these antibiotics give favorable results
in treatment of testicular tumors. It was shown that these antibiotics
exhibit side effects such as gastrointestinal, hepatic, renal and bone
marrow toxicities. The major clinically limiting toxicity of mithramycin
was a hemorrhagic diathesis associated with a precipitous thrombocytopenia.
It is of considerable interest that hemorrhagic diathesis
was not observed after administration of olivomycin I.

As olivomycin I possesses the best chemotherapeutic index among
the aureolic acid antibiotics, it can be considered as the best scaffold
for the development of novel semisynthetic aureolic acid analogs with
increased therapeutic indices and lower toxicity compared with the
parent antibiotic.
Here we describe chemical modifications of olivomycin I at the
2ў-keto group of the side chain of the aglycone moiety. The
antiproliferative and topoisomerase I (Topo-I)-poisoning activities
of the novel derivatives (2–7) were tested. One of the novel derivatives
showed pronounced antitumor activity in in vivo experiments on mice
bearing lymphocyte leukemia P-388, together with lower toxicity to
animals compared with the parent olivomycin I.

RESULTS

Chemistry
We developed a novel method of chemical modification of olivomycin
I (1) based on the introduction of a carboxyl group into the molecule
of the antibiotic. Reaction of olivomycin I (1) with carboxymethoxylamine
gave the key intermediate, 2ў-carboxymethoxime-olivomycin
I (2) (Scheme 1), which was further reacted with different amines in
the presence of PyBOP to give the corresponding amides 3–7. The
resulting compounds were purified by column chromatography on
silica gel.


Biological testing

The cytotoxicity of the compounds in comparison with the parent
olivomycin I was tested. Cells were incubated with drugs for 48–72 h
to ensure the completion of late events in cell death. Table 1 shows
the comparative potencies of these compounds against the
wild-type murine leukemia L1210 cells, the human leukemia cell
line K562 and the human malignant T-lymphocyte Molt4/C8 and
CEM cells.
All novel derivatives (2–7) caused cell death at higher concentrations
than olivomycin I. Remarkably, amides with the bulky
hydrophobic substituents (adamantyl- 5; tert-butyl-, 6) showed antiproliferative
activity that was at an IC50 of only one order of
magnitude higher (for L1210: 0.19 and 0.20 mM, correspondingly)
than that of olivomycin I, but at a markedly lower IC50 than that of
2ў-carboxymethoxime-olivomycin I (2) or the amides with small or
hydrophilic substituents 3, 4 and 7 (IC50 for L1210: 6.5–20 mM).
To identify tentative intracellular targets important for cytotoxicity
of olivomycin I and its novel derivatives (2–7), we tested these
compounds for their ability to modulate Topo-I activity in vitro.
Olivomycin I (1) and all novel derivatives (2–7) were potent Topo-I
inhibitors at all concentrations investigated (0.5–20 mM) (data not
shown). Figure 1 shows the results of electrophoretic analysis of the
relaxation products of the Topo-I-dependent supercoiled DNA relaxation
in the absence and presence of the antibiotics olivomycin I and 5.
In the absence of antibiotics (track of Topo-I), the reaction led to a set
of topoisomers and the disappearance of the supercoiled form of
DNA. This effect was revealed by the presence of residual amounts of
rapidly migrating topoisomers. Olivomycin I (1) inhibited Topo-I
activity at all concentrations investigated (0.5–20 mM); compound 5
was less active but still a potent Topo-I inhibitor—the rapidly
migrating topoisomers were observed on the track starting at the
compound concentration of 2.5 mM.

When 5 was i.p. injected to mice with leukemia P-388 72 h after i.p.
implantation of tumor, 62% increase of lifespan (ILS) at the dose
of 50mg kg was achieved.

Compound 5 did not show any cumulative toxic effects at the
quintuple doses of 5 and 10mg kg (25 and 50mg kg 1 total dose,
respectively), whereas all of the mice that received 1 (5 mg kg 1 daily)
had drug-related toxic death after the third injection (data no shown).
All mice that received daily doses of 5 and 10mg kg 1 had tumorrelated
death. The ILS was 43% compared with the group of untreated
mice (Figure 2). Quintuple injections of 5 at 20mg kg 1 per dose
(100mg kg total dose) resulted in drug-related death of 15% of the
mice.

Micafungin: a sulfated echinocandin

 

Fungal infections cause not only superficial diseases such as athlete’s
foot and onychomycoses, but also life-threatening diseases. Serious
deep-seated fungal infections caused by Candida spp., Aspergillus spp.
and Cryptococcus neoformans are a threat to human health. Incidences
of these systemic fungal infections have increased significantly over the
past few years. The major reasons for this dramatic increase are the
extensive use of broad-spectrum antibiotics and the growing number
of immunocompromised patients with acquired immunodeficiency
syndrome (AIDS), cancer and transplants.

In the mid 1900s, few compounds, such as polyenes (for example,
nystatin and amphotericin B) and flucytosine, were available for
antifungal chemotherapy. Although the development of azole drugs
started in the early 1970s, only a limited number of antifungal agents
were available for treatment of life-threatening fungal infections.
Moreover, the existing agents had disadvantages, such as the significant
nephrotoxicity of amphotericin B3 and the emergence of resistance
to the azoles.4 To overcome these defects, lipid formulations of
polyenes were developed to reduce toxicity, and new triazoles (for
example, voriconazole, ravuconazole and posaconazole) were developed
to improve the antifungal spectra or susceptibility to azoleresistant
isolates.5 Despite a number of therapeutic advancements,
there was a need to develop a new class of antifungal agents with novel
mechanisms of action.

The echinocandins were a new class of antifungal drugs developed
for the first time since azoles. The first launched echinocandin was
caspofungin acetate (Merck & Co. Inc. (Merck), Readington, NJ, USA),
followed by micafungin (Fujisawa Pharmaceutical Co., Ltd, now
Astellas Pharma Co., Ltd, Fujisawa, Japan) and anidulafungin (Vicuron
Pharmaceuticals Inc., now Pfizer Inc., New York, NY, USA), which was
originally developed by Eli Lilly and Company (Indianapolis, IN, USA)
(Lilly) as LY 303366 and subsequently licensed to Vicuron (formerly
Versicor) as VER-002. The approved echinocandins are synthetically
modified lipopeptides that originate from natural compounds produced
by filamentous fungi. The original anidulafungin, caspofungin
and micafungin compounds were echinocandin B from Aspergillus
nidulans var. echinulatus,6 pneumocandin B0 from Glarea lozoyensis7
and FR901379 from Coleophoma empetri,8 respectively.
Although natural echinocandins have potent antifungal activity
in vitro, their structures were chemically altered to improve their
absorption, distribution, metabolism and excretion characteristics.
Such operations were initiated by Lilly on echinocandin B to yield
cilofungin.9 This compound was subjected to Phase II clinical trials,
but was abandoned due to toxicity. Further modification of the
structure by converting the phenolic hydroxyl to a sodium phosphate
ester produced the more soluble prodrug LY307853, which resulted in
the active form, LY303366.10 Merck has produced MK-0991 using
pneumocandin B0 as the starting material.11 MK-0991 likewise possesses
increased water solubility. Other reviews on echinocandins or
individual antifungal agents have reported their usefulness in clinical
practice.12–16 This review describes the discovery and development of
micafungin, focusing on the chemical diversity of echinocandins.

DISCOVERY OF FR901379

The seed compounds of micafungin, FR901379 and two related
compounds (FR901381 and FR901382), were discovered at Fujisawa
Pharmaceutical Co., Ltd in 1989 from the screening of approximately
6000 microbial broth samples (Figure 1). These new compounds were
categorised as members of the echinocandin class of lipopeptides.
Echinocandin B, pneumocandin B0 and other echinocandin lipopeptides
are structurally characterized by a cyclic hexapeptide acylated
with a long side chain, and have an excellent anti-Candida activity
attributed to selective inhibition of 1,3-b-glucan synthesis, although
their intrinsic water insolubility is a major barrier for drug development.
17–19 However, FR901379 and related compounds showed both
high water solubility and a strong antifungal effect on Candida spp.20
The structural difference between FR901379 and the other echinocandins
is that FR901379 has a sulfate moiety in its molecule (Figure 1,
circled). This residue was speculated to be the basis for the high water
solubility of FR901379 (soluble in water even at a concentration of
50mgml 1, a concentration at which other compounds have low
solubility). To probe this hypothesis, FR901379 was digested with aryl
sulfatase from Aerobacter aerogenes (Table 1), after which the water
solubility of the desulfated molecule (FR133302) was decreased to
1mgml 1, even though the inhibitory activity on 1,3-b-glucan
synthase did not decrease markedly.21 This result indicated that the
excellent water solubility of FR901379 was attributed to the sulfate
moiety in its structure.

The IC50 value of FR901379 on 1,3-b-glucan synthase is
0.7 mgml1, which is superior to that of echinocandin B (Table 1).
The in vitro antifungal activity of FR901379 and related compounds
against both Candida albicans and A. fumigatus indicates a higher
potency than that of aculeacin A (Table 2); however, it is only weakly
active against A. fumigatus. None of these compounds show antifungal
activity against C. neoformans. Table 3 shows the therapeutic effect of
FR901379 in a murine C. albicans infection model in which drugs
were administered s.c. for four consecutive days. FR901379 and related
compounds significantly prolonged the survival of infected mice.
FR901379 was the most potent compound, with an ED50 value of
2.7mg kg1 14 days after the infection. This value was almost
comparable to that of fluconazole. In spite of its potent antifungal
activity and its good water solubility, FR901379 could not be developed
further because of class-specific reticulocyte lysis at low concentrations
(Table 4), although the lytic activity of FR901379 was
weaker than that of amphotericin B.
The producer strain of FR901379, identified as C. empetri F-11899,
was originally isolated from a soil sample collected at Iwaki City,
Fukushima Prefecture, Japan. Its morphological characteristics were
determined on the basis of cultures on sterilized azalea leaf affixed to a
Miura’s LCA plate, because the strain produced conidial structures on
the leaf segment alone.

30 years since the discovery of staurosporine

 

The discovery of medically useful natural products has heralded
hitherto unimagined possibilities in the chemotherapy of human
and animal diseases.

It is well known that important medical compounds, such as
penicillin, cyclosporine A and lovastatin, were only developed as
drugs once their key properties were recognized, more than 10 years
after their initial discovery.4 Similarly, in the case of staurosporines,
their crucial protein kinase inhibitory properties were only identified a
decade or so after their initial discovery.
In 1986, 9 years after the isolation of staurosporine from a streptomyces,
the related natural indolocarbazole products, staurosporine and
K252, were shown to be nanomolar inhibitors of protein kinases, offering
tremendous promise for drug development. The reports led many
pharmaceutical companies to begin searching for selective protein kinase
inhibitors through natural product screening and chemical synthesis,
with the result that, during the 1990s, protein kinases became the second
most important drug target after G-protein-coupled receptors.

In parallel with the development of indolocarbazoles as
anticancer drugs targeting protein kinases, mammalian DNA
topoisomerase I was shown to be a new target for indolocarbazoles
by Yamashita et al. Thereafter, many antitumor indolocarbazoles
have been synthesized, as DNA topoisomerases were known to be
targets for antitumor drugs such as camptothecin and VP-16.
DNA topoisomerases alter DNA topology by transiently breaking
and re-sealing one strand of DNA through a covalent protein–DNA
intermediate. In 1996, it was shown that topoisomerase I has an
intrinsic protein kinase activity (Topo I kinase) required for phosphorylation
of the SR (serine arginine-rich) protein required for
splicing.
The action of indolocarbazole derivatives on topoisomerase
indicated that these compounds may selectively interact with ATPbinding
sites of not only protein kinases but also other proteins. As an
example of this, during the current decade, it was shown that ABCG2,
an ABC transporter with importance in drug resistance, oral drug
absorption and stem cell biology, could be a key new target for
indolocarbazoles.
This review outlines the pivotal pioneering studies relating to the
discovery, biosynthesis and biological activities of natural indolocarbazole
products.

PRODUCING ORGANISM

Staurosporine was discovered in 1977 in a culture of an actinomycete
(Streptomyces strain AM-2282T) while screening for microbial alkaloids
using chemical detection methods11. The strain AM-2282T
(NRRL 11184, ATCC 55006) has been renamed through repeated
revisions of the taxonomy of soil Actinomyces as Streptomyces staurosporeus
AM-2282T in 1977, Saccharothrix aerocolonigenes subsp.
staurosporea AM-2282T in 199512 and Lentzea albida in 2002. Over
the past 30 years, staurosporine and related natural indolocarbazole
compounds have been isolated from several actinomycetes (including
Streptomyces, Saccharothrix, Lentzea, Lechevalieria, Nocardia, Nocardiopsis,
Nonomuraea, Actinomadura and Micromonospora) as well as
from myxomycetes (slime molds) and cyanobacteria (Figure 1).
Staurosporine derivatives have also been isolated from marine
invertebrates, such as sponges, tunicates, bryozoans and mollusks.
However, it remains unknown whether invertebrates actually have
genes for indolocarbazole biosynthesis, as many natural products from
marine invertebrates are produced by associated microorganisms.14
Interestingly, half of the 14 indolocarbazole-producing strains
deposited in the global culture collection have been isolated from
Japanese soils. In the 1980 s, fermentation broths of 5163 new Japanese
soil isolates were tested and five Streptomyces were found to produce
staurosporine, together with new analogs (UCN-01 and UCN-02
(stereo-isomers of 7-hydroxy staurosporines)). In other words, ca
0.1% of newly isolated soil actinomycetes were shown to produce
staurosporine using a fixed culture condition.15
In 1993, staurosporine and K252a were shown to inhibit in vitro
phosphorylation of crude extracts from Streptomyces griseus and also
from a staurosporine-producing Streptomyces sp.16 Although staurosporine
did not show significant antibacterial activity, it was shown to
affect cell differentiation processes in Streptomycetes, such as pigment
production and spore formation, depending on the AfsK family
serine/threonine protein kinases involved. Later, on the basis of
genome sequence analysis of Streptomyces avermitilis in 200117
and Streptomyces coelicolor in 2002,18 it was revealed that more than
30 protein kinase genes are coded in these organisms. Further research
is needed to determine the exact role and impact of staurosporine on
differentiation of producing strains and microorganisms in soil.

BIOSYNTHESIS OF STAUROSPORINE

Biosynthetic studies carried out in the 1980s and 1990s using isotopelabeled
precursors showed that the indolocarbazole structure of
staurosporine is derived from two molecules of tryptophan, and
that the sugar moiety is derived from glucose and methionine.
Cloning of the biosynthetic genes of staurosporine was triggered in
2000 by identification of the ngt gene encoding N-glycosyltransferase.
Ohuchi et al.19 heterologously expressed the ngt transferase gene from
Lechevalieria aerocolonigenes, a rebeccamycin producer, in Streptomyces
lividans and showed that ngt is responsible for N-glycosylation of the
indolocarbazole chromophore. Starting from the ngt gene, whole
biosynthetic gene clusters of staurosporine and rebeccamycin have
been cloned by Onaka et al.20,21 and Sanchez et al.22 To date, structures
of the accumulated products from 18 single-gene disruption mutants
of staurosporine and rebeccamycin biosynthesis gene clusters have
been identified (Figure 2).
Studies of these accumulated products and the gene function
predicted by the amino-acid sequence database searches have revealed
the biosynthetic pathway of staurosporine and rebeccamycin.14,23
(Figure 3).
In staurosporine biosynthesis, staO initiates synthesis by catalyzing
L-tryptophan to the imine form of indole-3-pyruvic acid (IPA imine)
and staD, and then catalyzes the coupling of two IPA imines to yield
chromopyrrolic acid. Formation of the indolocarbazole core of
staurosporine is catalyzed by staP, which converts chromopyrrolic
acid into three indolocarbazole compounds, staurosporine aglycone
(K252c), 7-hydroxy-K252c and acryriaflavin A, by intramolecular C–C
bond formation and oxidative decarboxylation. Crystallography of
P450 staP revealed that a heme of staP removes two electrons from the
indole ring to generate an indole cation radical, and intramolecular
radical coupling then forms the C–C bond to yield the indolocarbazole
core.24 The presence of staC predominantly directs the formation
of K252c. staG catalyzes N-glycosidic bond formation between N-13
and C-6ў and then staN, a P450 homolog, catalyzes an additional
C–N bond formation between N-12 and C-2ў. These two enzymes
convert K252c to 3ў-O-demethyl, 4ў-N-demethyl-staurosporine
through holyrine A and holyrine B.

The genes involved in the main pathway of indolocarbazole
structure formation in staurosporine and rebeccamycin showed striking
similarity between staO, staD, staP, staC and staG, and rebO, rebD,
rebP, rebC and rebG, respectively. The formation of chromopyrrolic
acid or 11,11-dichlorochromopyrrolic acid, key intermediates of
indolocarbazole biosynthesis, is catalyzed by staD or rebD. It is
noteworthy that the staD family includes only two homologs, rebD
and VioB, which are involved in violacein biosynthesis. The staD
family is a new type of hemoprotein with a novel structure and
function.

N-glycosidic bond formation between the N-12 and C-1ў positions
is catalyzed by staG or rebG N-glycosyltransferases. rebG is the same
gene that was cloned in 2000 by Ohuchi et al. as ngt, which can
catalyze the N-glycosylation of the indolocarbazole chromophore.
In the staurosporine structure, there exists an additional, unusual
C–N bond between the N-13 and C-6ў positions. Onaka et al. showed
through gene disruption and bioconversion experiments that staN, a
P450 homolog, is responsible for this unusual C-N bond formation.
StaN was the first example used to show that the P450 homolog is
involved in N-glycosidic bond formation. Deletion of staG abolished
glycosylation and led to accumulation of K252c, whereas deletion of
staN resulted in the production of holyrine A. Salas et al. also showed
the function of staN in C–N bond formation by heterologous
expression of the staN gene.

Herbicides

 

Herbicides are chemicals marketed to inhibit or interrupt normal
plant growth and development. They are widely used in agriculture,
industry and urban areas for weed management. Approximately
30 000 kinds of weeds are widely distributed in the world; yield losses
caused by 1800 kinds of weeds are approximately 9.7% of total crop
production every year.98 Herbicides provide cost-effective weed control
with a minimum of labor. Most are used on crops planted in large
acreages, such as soy, cotton, corn and canola. There are numerous
classes of herbicides with different modes of action, as well as different
potentials for adverse effects on health and the environment. Over the
past century, chemical herbicides, used to control various weeds, may
have caused many serious side effects, such as injured crops, threat to
the applicator and others exposed to the chemicals, herbicide-resistant
weed populations, reduction of soil and water quality, herbicide
residues and detrimental effects on non-target organisms.100 For
example, alachlor and atrazine were reported to cause cancer in
animal tests. With increasing global environmental consciousness,
bioherbicides, which are highly effective for weed control and environmentally
friendly as well, are very attractive both for research and
for application. Microbial herbicides can be divided into microbial
preparations (microorganisms that control weeds) and microbially
derived herbicides.
The first microbial herbicide was independently discovered in
Germany and Japan. In 1972, the ZaЁhner group in Germany isolated
phosphinothricin tripeptide, a peptide antibiotic consisting of two
molecules of L-alanine and one molecule of the unusual amino acid
L-phosphinothricin; that is, N(4[hydroxyl(methyl)phosphinoyl]homoalanyl)
alanylalanine. They isolated it from Streptomyces viridochromogenes
as a broad-spectrum antibacterial including activity against
Botrytis cinerea. In Japan, it was discovered at the Meiji Seiki
laboratories in 1973 from S. hygroscopicus and named bialaphos.102
The bioactive L-phosphinothricin is a structural analog of glutamic
acid, acting as a competitive inhibitor of glutamine synthetase, and has
bactericidal (Gram-positive and Gram-negative bacteria), fungicidal
(B. cinerea) and herbicidal properties. Glufosinate (DL-phosphinothricin)
(without Ala-Ala) was developed as a herbicide. Therefore, the
agent acts as a herbicide with or without Ala-Ala. Bialaphos has no
influence on microorganisms in the soil and is easily degraded in the
environment, having a half-life of only 2 h. This low level of environmental
impact is of great interest to environmentalists.
Antiparasitics and ruminant growth stimulants
In 2006, the global animal health market was valued at US$16 billion,
of which 29% was derived from parasiticides. Parasites are organisms
that inhabit the body and benefit from a prolonged, close association
with the host. Antiparasitics are compounds that inhibit the growth or
reproduction of a parasite; some antiparasitics directly kill parasites. In
general, parasites are much smaller than their hosts, show a high
degree of specialization for their mode of life and reproduce more
quickly and in greater numbers than their hosts. Classic examples of
parasitism include the interactions between vertebrate hosts and such
diverse animals as tapeworms, flukes, Plasmodium species and fleas.
Parasitic infections can cause potentially serious health problems
and even kill the host. Parasites mainly enter the body through
the mouth, usually through ingestion of tainted food or drink. This
is a very common problem in tropical areas, but is not limited to
those regions. There are 3200 varieties of parasites in four major

categories: Protozoa, Trematoda, Cestoda and Nematoda. The major
groups include protozoans (organisms having only one cell) and
parasitic worms (helminths). Each of these can infect the digestive
tract, and sometimes two or more can cause infection at the same
time. The WHO reported that approximately 25% of the world’s
population is infected with roundworms. In addition, a major
agricultural problem has been the infection of farm animals by worms.
The predominant type of antiparasitic screening effort over the
years was the testing of synthetic compounds against nematodes, and
some commercial products did result. Certain antibiotics were also
shown to possess antihelmintic activity against nematodes or cestodes,
but these failed to compete with the synthetic compounds. Although
Merck had earlier developed a commercially useful synthetic product,
thiabendazole, they had enough foresight to examine microbial
broths for antihelmintic activity, and found a non-toxic fermentation
broth that killed the intestinal nematode Nematosporoides dubius in
mice. The Streptomyces avermitilis culture, isolated by OЇ mura and
coworkers at the Kitasato Institute in Japan, produced a family of
secondary metabolites (eight compounds) with both antihelmintic
and insecticidal activities. These compounds, named ‘avermectins,’
are pentacyclic, 16-membered macrocyclic lactones, that harbor a
disaccharide of the methylated sugar, oleandrose, with exceptional
activity against parasites, especially Nemathelminthes (nematodes)
and arthropod parasites (10 times higher than any known synthetic
antihelmintic agent). Surprisingly, avermectins lack activity against
bacteria and fungi, do not inhibit protein synthesis and are
not ionophores. Instead, they interfere with neurotransmission in
many invertebrates, causing paralysis and death by neuromuscular
attacks.
The annual market for avermectins surpasses US$1 billion. They are
used against both nematode and arthropod parasites in sheep, cattle,
dogs, horses and swine. A semisynthetic derivative, 22,23-dihydroavermectin
B1 (‘ivermectin’) is 1000 times more active than thiabendazole
and is a commercial veterinary product. The efficacy of
ivermectin has made it a promising candidate for the control of
human onchocerciasis and human strongyloidiasis. Another avermectin,
called doramectin (or cyclohexyl avermectin B1), produced by
‘mutational biosynthesis’ was commercialized for use by food animals.
107 A semisynthetic monosaccharide derivative of doramectin
called selamectin is the most recently commercialized avermectin, and
is active against heartworms (Dirofilaria immitis) and fleas in companion
animals. Although the macrocyclic backbone of each of these
molecules (ivermectin, doramectin and selamectin) is identical, there
are different substitutions at pharmacologically relevant sites such as
C-5, C-13, C-22,23 and C-25.108 The avermectins are closely related to
the milbemycins, a group of non-glycosidated macrolides produced by
S. hygroscopicus subsp. Aureolacrimosus. These compounds possess
activity against worms and insects.
Coccidiostats are used for the prevention of coccidiosis in both
extensively and intensively reared poultry. Coccidiosis is the name
given to a common intestinal disease caused by the invading protozoan
parasites of the genus Eimeria that affects several different animal
species (cattle, dogs, cats, poultry, etc.). The major damage is caused
by the rapid multiplication of the parasite in the intestinal wall and the
subsequent rupture of the cells of the intestinal lining, leading to high
mortality and severe loss of productivity. Coccidia are obligate
intracellular parasites that show host specificity; only cattle coccidia
will cause disease in cattle; other species-specific coccidia will not.
For many years, synthetic compounds were used to combat
coccidiosis in poultry; however, resistance developed rapidly. A solution
came on the scene with the discovery of the narrow-spectrum
polyether antibiotic monensin, which had extreme potency against the
coccidian. Made by Streptomyces cinnamonensis, monensin led the
way for additional microbial ionophoric antibiotics, such as lasalocid,
narasin and salinomycin. All are produced by various Streptomyces
species. They form complexes with the polar cations K+, Na+, Ca2+
and Mg2+, severely affecting the osmotic balance in the parasitic cells
and thus causing their death. The widespread use of anticoccidials
has revolutionized the poultry industry by reducing the mortality and
production losses caused by coccidiosis. Of great interest was another
extremely valuable application of monensin; that is, growth promotion
in ruminants. Synthetic chemicals had been tested for years to
inhibit wasteful methane production by cattle and sheep and increase
fatty acid formation (especially propionate) to improve feed efficiency;
however, they failed. The solution was monensin, which became a
major success as a ruminant growth enhancer.
For more than 40 years, certain antibiotics have been used in foodanimal
production to enhance feed utilization and weight gain.112
From a production standpoint, feed antibiotics have been consistently
shown to improve animal weight gain and feed efficiency, especially in
younger animals. These responses are probably derived from an
inhibitory effect on the normal microbiota, which can lead to reduced
intestinal inflammation and improved nutrient utilization.113 Pigs
in the USA are exposed to a great variety of antibiotics. These include
b-lactam antibiotics (including penicillins), lincosamides and macrolides
(including erythromycin and tetracyclines). All these groups have
members that are used to treat infections in humans. In addition,
bacitracin, flavophospholipol, pleuromutilins, quinoxalines and virginiamycin
are utilized as growth stimulants. Flavophospholipol and
virginiamycin are also used as growth promoters in poultry.
As described above, cattle are also exposed to ionophores such as
monensin to promote growth. The Animal Health Institute of
America114 has estimated that without the use of growth-promoting
antibiotics, the USA would require an additional 452 million chickens,
23 million more cattle and 12 million more pigs to reach the levels of
production attained by the current practices.
Considering that animal health research and the development of
new anti-infective product discovery have decreased, the discovery of
new antibiotics has decreased over the past 15 years, with few new
drug approvals.115 Therefore, it will be incumbent on veterinary
practitioners to use the existing products in a responsible manner to
ensure their longevity. It remains to be seen what effects the dearth of
new antibiotics for veterinary medicine will have on the future
practice of veterinary medicine, production agriculture, food safety
and public health.
Since the 1999 EU decision to prohibit antibiotic use for foodanimal
growth promotion, four antibiotic growth promoters have
been banned, including the macrolide drugs tylosin and spiramycin.
117 Although macrolides are no longer formally used as ‘growth
promoters,’ their use under veterinary prescription has risen from 23
tons in 1998 to 55 tons in 2001, which suggests that more of them are
being used now than before the prohibition.

Insecticides

 

An insecticide is a pesticide used against insects in all developmental
forms. They include ovicides and larvicides used against the eggs and
larvae of insects, respectively. Insecticides are used in agriculture,
medicine, industry and households. The use of insecticides is believed
to be one of the major factors behind the increase in agricultural
productivity in the twentieth century.
Synthetic insecticides pose some hazards, whereas natural insecticides
offer adequate levels of pest control and pose fewer hazards.
Microbially produced insecticides are especially valuable because their
toxicity to non-target animals and humans is extremely low.
Compared with other commonly used insecticides, they are safe for
both the pesticide users and consumers of treated crops. The action of
microbial insecticides is often specific to a single group or species of
insects, and this specificity means that most microbial insecticides do
not naturally affect beneficial insects (including predators or parasites
of pests) in treated areas.
The spinosyns (A83543 group) are a group of natural products
produced by Saccharopolyspora spinosa that were discovered in 1989.
The researchers isolated spinosyn A and D, as well as 21 minor
analogs. They are active on a wide variety of insect pests, especially
lepidopterans and dipterans, but do not have antibiotic activity.95 The
compounds attack the nervous system of insects by targeting two key
neurotransmitter receptors, with no cross-resistance to other known
insecticides. The spinosyns are a family of macrolides with 21 carbon
atoms, containing four connected rings of carbon atoms at their core
to which two deoxysugars (forosamine and 2,3,4, tri-O-methylrhamnose,
which are required for bioactivity) are attached. Novel
spinosyns have been prepared by biotransformation, using a genetically
engineered strain of Saccharopolyspora erythraea.96 A mixture of
spinosyn A (85%) and D (15%) (spinosad) is being produced through
fermentation and was introduced to the market in 1997 for the control
of chewing insects on a variety of crops. Spinosyn formulations were
recently approved for use on organic crops and for animal health
applications.
Recently, a new naturally occurring series of insect-active compounds
was discovered from a novel soil isolate, Saccharopolyspora
pogona NRRL30141. The culture produced a unique family of
over 30 new spinosyns. They have a butenyl substitution at the 21
position on the spinosyn lactone and are named butenyl-spinosyns
or pogonins.

Hypocholesterolemic drugs

 

Atherosclerosis is generally viewed as a chronic, progressive disease
characterized by the continuous accumulation of atheromatous plaque
within the arterial wall. The past two decades have witnessed the
introduction of a variety of anti-atherosclerotic therapies. The statins
form a class of hypolipidemic drugs used to lower cholesterol by
inhibiting the enzyme HMG-CoA reductase, the rate-limiting enzyme
of the mevalonate pathway of cholesterol biosynthesis. Inhibition of
this enzyme in the liver stimulates low-density lipoprotein (LDL)
receptors, resulting in an increased clearance of LDL from the bloodstream
and a decrease in blood cholesterol levels. Through their
cholesterol-lowering effect, they reduce the risk of cardiovascular
disease, prevent stroke and reduce the development of peripheral
vascular disease. In addition, they are anti-thrombotic and antiinflammatory.

Nowdays there are a number of statins in clinical use. The entire
group of statins reached an annual market of nearly US$30 billion
before it became a generic pharmaceutical. The first member of the
group (compactin; mevastatin) was isolated as an antibiotic product
of Penicillium brevicompactum and later from Penicillium citrinum.
Although not of commercial importance, compactin’s derivatives
achieved overwhelming medical and commercial success. An ethylated
form, known as lovastatin (monacolin K; mevinolin), was isolated in
the 1970s in the broths of Monascus ruber and Aspergillus terreus.91
Lovastatin, the first commercially marketed statin, was approved
by the FDA in 1987. A semisynthetic derivative of lovastatin is
simvastatin, a major hypocholesterolemic drug, selling for US$7
billion per year before becoming generic. Another statin, pravastatin
(US$3.6 billion per year), is made through different biotransformation
processes from compactin by Streptomyces carbophilus92 and Actinomadura
sp.93 Other genera involved in the production of statins are
Doratomyces, Eupenicillium, Gymnoascus, Hypomyces, Paecilomyces,
Phoma, Trichoderma and Pleurotus.94 A synthetic compound, modeled
from the structure of the natural statins, is atorvastin, which has been
the leading drug of the entire pharmaceutical industry in terms of
market share (approximately US$14 billion per year) for many years.